Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

• Published in: The Journal of biological chemistry 2013-10, Vol.288 (43), p.31030-31041
• Main Authors: Mangialavori, Irene C., Ferreira-Gomes, Mariela S., Saffioti, Nicolás A., González-Lebrero, Rodolfo M., Rossi, Rolando C., Rossi, Juan Pablo F.C.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; ATP; Calcium; Calcium ATPase; Calcium Transport; Erythrocyte Membrane - chemistry; Erythrocyte Membrane - enzymology; Humans; Ion Transport - physiology; Membrane Biology; Models, Chemical; Phosphatidylcholines - chemistry; Phosphatidylcholines - metabolism; Plasma Membrane Calcium-Transporting ATPases - chemistry; Plasma Membrane Calcium-Transporting ATPases - metabolism; Protein Conformation; Protein Structure, Tertiary; Reaction Cycle; Vanadate
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.494633

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only. Background: The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes. Results: We identified different conformations after the association of Ca2+, ATP, and vanadate to PMCA. Conclusion: PMCA forms a stable complex with Ca2+ and vanadate; ATP can bind to all pump conformations. Significance: This study found a new intermediate in the PMCA reaction cycle; all of the intermediates interact with ATP.